Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates

ABSTRACT

Disclosed are methods, systems, components, and compositions for cell-free synthesis of proteins and glycoproteins. The methods, systems, components, and compositions may be utilized for incorporating non-standard amino acids (nsAAs) into cell-free synthesized proteins and glycosylating or otherwise modifying the cell-free synthesized proteins in vitro. The nsAAs of the cell-free synthesized protein may be modified via glycosylation or other modification.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application represents the U.S. national stage entry ofInternational Application PCT/US2019/027733, filed Apr. 16, 2019, whichclaims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/658,181, filed on Apr. 16, 2018, thecontent of which is incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under MCB1413563 awardedby the National Science Foundation. The government has certain rights inthe invention.

BACKGROUND

The field of the invention relates to in vitro synthesis of proteins. Inparticular, the field of the invention relates to one-pot systems forincorporating non-standard amino acids and glycosylation into cell-freesynthesized proteins.

Cell-free protein synthesis (CFPS) using extracts from prokaryoticsource strains such as E. coli has undergone a transformational shiftfrom an exploratory platform used in the discovery of the genetic codeto a present-day, high-yielding protein production platform [1]. Thisshift is fueled by the open nature of this system, allowing for rapidcombination, supplementation, and optimization of the physiochemicalenvironment for increasing protein yields and batch reaction duration[2, 3]. Now, cell-free systems are seen as a complement to in vivoprotein expression and can be used as both a prototyping platform due toits simplicity, easiness, and modular design for protein expression[4-6] as well as a large-scale production platform for difficult toexpress proteins in vivo [7]. The transition from exploratory platformto high-yielding protein production platform has come about, at least inpart, by complex strain engineering to stabilize biological substratesin the cell-free reaction mixtures [8, 9]. These genetic modificationstargeted the deletion of proteins known to affect the stability of DNA[10], mRNA [8, 11], protein [12], energy [13], and amino acids [14, 15]in the cell-free reaction. In addition to strain engineering efforts,activation of multiple biological pathways [16], decreases in cost [17],and improved understanding of reaction contents makes CFPS an attractiveplatform for the production of new kinds of high-value proteins.

One area of great interest for the application of cell-free systems isthe production of modified proteins containing non-standard amino acids.Incorporating non-standard amino acids or unnatural amino acids (nsAAs)allows for the production of proteins with novel structures andfunctions that are difficult or impossible to create using the 20canonical amino acids [18, 19]. Recently, cell-free protein synthesis(CFPS) systems have been employed to increase yields of proteins bearingnsAAs [20, 21], achieve direct protein-protein conjugation [22], exploredrug discovery [23], and enhance enzyme activity [24, 25].

Typically, nsAA incorporation systems use amber suppression technologyto insert nsAAs into proteins, a method by which an in-frame amber (TAG)stop codon is utilized as a sense codon for assigning nsAAs [26, 27].Amber suppression technology, however, has limited efficiency for nsAAincorporation because of the presence of release factor 1 (RF1). RF1naturally binds the amber stop codon (TAG) [28] and prematurelyterminates protein translation. Methods to counteract this competitivetermination of the TAG stop codon include increasing the addition ofcompeting tRNA [21], tagging and purifying out RF1 [29], release factorengineering [30], and genomically recoding strains to remove RF1 andreassigning all occurrences to the synonymous TAA codon [31]. High-yieldprotein production with multiple-site incorporation of NSAAs stillremains a critical challenge.

Glycosylation is possible in some CFPS systems. The development of ahighly active E. coli CFPS platform has prompted recent efforts toenable glycoprotein production in E. coli lysates through the additionof orthogonal glycosylation components. In one study, Guarino and DeLisademonstrated the ability to produce glycoproteins in E. coli CH'S byadding purified lipid-linked oligosaccharides (LLOs) and the C. jejuniOST to a CFPS reaction. Yields of between 50-100 μg/mL of AcrA, a C.jejuni glycoprotein, were achieved [64]. Despite these recent advances,bacterial cell-free glycosylation systems have been limited by theirinability to co-activate efficient protein synthesis and glycosylation.We recently developed a cell-free glycoprotein synthesis (CFGpS) systemthat addresses this limitation by enabling modular, coordinatedtranscription, translation, and N-glycosylation of proteins in E. colilysates selectively enriched with glycosylation enzymes (see WO2017/117539, the content of which is incorporated herein by reference inits entirety). Here, we demonstrate in vitro incorporation ofnon-standard amino acids that include a chemical moiety forglycosylation or other post-translational modifications in crude celllysates.

SUMMARY

Disclosed are methods, systems, components, and compositions forcell-free synthesis of proteins and glycoproteins. The methods, systems,components, and compositions may be utilized for incorporatingnon-standard amino acids (nsAAs) into cell-free synthesized proteins andglycosylating or otherwise modifying the cell-free synthesized proteinsin vitro. The nsAAs of the cell-free synthesized protein may be modifiedvia glycosylation or other modification.

In some embodiments, the methods, systems, components, and compositionsrelate to genomically recoded and engineered organisms. The disclosedgenomically recoded and engineered organisms may be used to prepareextracts for platforms and methods for preparing sequence definedbiopolymers, proteins, or glycoproteins in vitro. In particular, themethods, systems, components, and compositions relate to genomicallyrecoded and engineered organisms comprising a strain which isgenetically modified in a manner selected from: (i) modified to bedeficient in release factor 1 (RF-1) or a genetic homolog thereof; (ii)modified to be deficient in one or more of endA and gor; (iii) modifiedto be deficient in one or more of gmd and waal; (iv) modified to produceglycosylation components such as lipid-linked oligosaccharides (LLOs),oligosaccharyltransferases (OSTs) or both of LLOs and OSTs; (v) modifiedto express an orthogonal translation system(s) (OTS) including one ormore of orthogonal tRNA(s), engineered aminoacyl-tRNA synthetases(s)(aaRS), engineered elongation factors (EF), or any combination of theorthogonal tRNAs, the engineered aaRSs, and the engineered EFs.

In other embodiments, the methods, systems, components, and compositionsrelate to a platform for preparing a sequence defined biopolymer,protein, or glycoprotein in vitro, the platform comprising a cellularextract from the genomically recoded and engineered organisms disclosedherein. In certain embodiments, a cellular extract prepared from thestrain of the genomically recoded and engineered organism is capable ofpreparing a sequence defined biopolymer, protein, or glycoproteinutilizing coupled in vitro translation/glycosylation in greater yieldand/or purity than previously disclosed strains.

In some embodiments, the platform further comprises an orthogonaltranslation system component configured to incorporate unnatural,nonstandard, and/or non-canonical amino acids. Suitable unnatural,nonstandard, and/or non-canonical amino acids may include, but are notlimited to, amino acids comprising a moiety that reacts and conjugateswith a corresponding moiety on a carbohydrate monomer (e.g., acarbohydrate monomer of a glycan) such as p-azido-L-phenylalanine (pAzF)or p-proparglyoxy-L-phenylalanine (pAcF). In certain embodiments, theorthogonal translation system component is expressed from a plasmidpresent in the genomically recoded organism, expressed from anintegration site in the genome of the genetically recoded organism,co-expressed from both a plasmid present in the genomically recodedorganism and an integration site in the genome of the geneticallyrecoded organism, expressed in an in vitro transcription and translationreaction, or added exogenously. In some embodiments, the cellularextract from the genomically recoded organism is a component in areaction mixture.

In some embodiments, the sequence defined biopolymer, protein, orglycoprotein prepared from the disclosed platforms and methods includesat least one unnatural amino acid that is modified to include a chemical“handle” or “moiety” (e.g., such as an azido or proparglyoxyl handle ormoiety), which provides an attachment point for a glycan via anoligosaccharyltransferase of via a chemical reaction with a glycan. Insome embodiments, the sequence defined biopolymer or protein encodes atherapeutic product, a diagnostic product, a biomaterial product, anadhesive product, a biocomposite product, or an agricultural product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Examples of tumor-associated glycans from Pochechueva et al.,Metabolites (2012) December; 2(4): 913-939. Illustrated are some of theglycans known to be involved in gynecological cancers.

FIG. 2 . Illustrative glycans observed in Chinese Hamster Ovary (CHO)cells from Sheridan et al., Nat. Biotechnol. (2007), volume 25, pages145-146.

FIG. 3 . Illustration of bacterial engineering enabling programmableglycosylation in Escherichia coli. (A) Illustration of Campylobacterjejuni N-glycosylation from Wacker et al., Science (2002), Nov. 29;298(5599):1790-3. (B) Illustration of Eukaryotic trimannose coreN-glycosylation from Valderrama-Rincon et al., Nat. Chem. Biol. (2012),Mar. 25; 8(5):434-6. doi: 10.1038/nchembio.921.

FIG. 4 . Illustration of the use of cell-free biology to enable precisecontrol over protein synthesis and glycosylation.

FIG. 5 . Crude E. coli lysates can be enriched with glycosylationcomponents via overexpression in the source strain for lysateproduction. (A) CjLLO plasmid which expresses Campylobacter jejunilipid-linked oligosaccharides (LLO) and CjOST plasmid which expressesCampylobacter jejuni oligosaccharide transferases (OST) are introducedinto a source strain to provide a cell lysate enriched in LLO and OST.An acceptor plasmid expressing an acceptor polypeptide is introducedinto a cell-free glycoprotein synthesis (CFGpS) reaction mixturecomprising the cell lysate to prepare a glycosylated acceptorpolypeptide. (B) Western blot of glycosylated single chain antibodyvariable fragment (scFv) and glycosylated green fluorescent protein(GFP) probed with anti-histidine antibody (α-His) or anti-glycanantibody (α-Glycan).

FIG. 6 . Western blot illustrating that proteins such as a single chainantibody variable fragment (scFv) can be glycosylated with bacterial andeukaryotic glycans using a cell-free glycoprotein synthesis (CFGpS)platform.

FIG. 7 . Four proteins approved by the US Food and Drug Administration(FDA) as adjuvant carriers for glycan antigens including Neisseriameningitides outer membrane protein (PorA), Tetanus toxoid ofClostridium tetani, Diptheria toxoid of Corynebacterium diptheriae,Non-typeable Haemophilus influenzae derived protein D (hpd).

FIG. 8 . Protein carriers can be glycosylated with Franciscellatularensis O antigen. (A) Crude E. coli lysates can be enriched withglycosylation components via overexpression in the source strain forlysate production. FtLLO plasmid which expresses Franciscella tularensislipid-linked oligosaccharides (LLO) including glycans forming theFranciscella tularensis O antigen, and CjOST plasmid which expressesCampylobacter jejuni oligosaccharide transferases (OST) are introducedinto a source strain to provide a cell lysate enriched in LLO and OST.An acceptor plasmid expressing an FDA-approved protein carrier isintroduced into a cell-free glycoprotein synthesis (CFGpS) reactionmixture comprising the cell lysate to prepare a glycosylated proteincarrier. (B) Western blot of glycosylated carrier proteins (PD, PorA,CRM197, TTlight) probed with anti-histidine antibody (α-His) oranti-Francisella tularensis O polysaccharide antibody (α-Ft O-PS).

FIG. 9 . Schematic illustration of the use of amber suppression in arelease factor 1 (RF-1) deficient strain to incorporate a non-standardamino acid (nsAA). (See Perez, Stark, and Jewett, Cold Spring HarborPerspect. Biol. (2016), Dec. 1; 8(12). pii: a023853. doi:10.1101/cshperspect.a023853). Orthogonal transfer RNA (o-tRNA)comprising the AUC anti-codon is charged with the nsAA via an orthogonalaminoacyl-tRNA synthetase (o-aaRS). Elongation factor thermos unstable(EF-Tu) then facilitates the selection and binding of the charged o-tRNAto the A-site of the ribosome and the nsAA is incorporated in thenascent peptide chain at the position of the amber (UAG) codon. Becausethe strain is RF-1 deficient, translation is not terminated at the ambercodon.

FIG. 10 . (Top) Illustration of combined incorporation of a non-standardamino acid (symbolized by star) and glycan (symbolized by structure ofsquares and circles) into a protein chain. (Bottom) Western blot ofprotein having an incorporated non-standard amino acid (nsAA) andglycosylation probed with anti-His antibody (α-His) or anti-glycanantibody (α-Glycan). aaRS designates the molecule weight of proteinchain having incorporated nsAA.

FIG. 11 . Derivatives of the E. coli 705 strain were created that weredeficient in waaL or deficient in both of waaL and gmd. (A) Analysisconfirming ΔwaaL deletion and combination of ΔwaaL and Δgmd deletions in705 strain. (B) Active sfGFP produced in cell-free protein synthesis(CFPS) reactions using cell lysates from the indicated strains. (C)Active sfGFP produced versus time in CFPS reactions using cell lysatesfrom the 705ΔwaaL strain and the 705ΔgmdΔwaaL strain.

FIG. 12 . Incorporation of para-azido-phenylaline (pAzF) into the sfGFPT216X protein via cell-free protein synthesis. A cell-free extract wasprepare from the 705ΔwaaL strain and a purified orthogonal amino-acyltRNA synthetase (aaRS) from Methanococcus jannaschii was added atincreasing amounts to prepare CFPS reaction mixtures.

FIG. 13 . Western blot illustrating coordinated amber suppression andglycosylation in cell-free glycoprotein synthesis (CFGpS) of testproteins (scFV13-R4, sfGFP-T216X, and sfGFP-E132X). A cell-free extractwas prepared from the 705ΔwaaL strain and test conditions included:addition of para-azido-phenylalanine and orthogonal amino-acyl tRNAsynthetase; addition of a cell free lysate comprising theoligosaccharide transferase from C. jejuni; addition of a cell freelysate comprising the oligosaccharide transferase from C. c; addition ofa cell free lysate comprising lipid-linked oligosaccharides from C.jejuni; and presence of the glycosylated sequon DQNAT ornon-glycosylated variant AQNAT in the synthesized protein.

FIG. 14 . Site-specific incorporation of a click-active ncAA into targetprotein using E. coli systems. (A) Schematic of the OTS used tosite-specifically install ncAAs. (B) In-gel fluorescence image ofSDS-PAGE gel demonstrating installation of the ncAA(para-azido-phenylalanine) into a 23 kDa target protein. Gel showsbiorthogonal labeling of the target protein with a fluorophore viaSPAAC.

FIG. 15 . CFGpS lysates enriched with o-tRNA are active for ncAAincorporation and for efficient glycosylation (A) CFPS yields of sfGFPencoded by plasmids containing 0, 1, 2, or 5 amber stop codons in CFGpSlysates enriched with bacterial LLO variants and containing allcomponents of the pAzF-OTS (B) Lysates containing amber suppression andglycosylation machinery efficiently, glycosylate a model proteinconstruct (sfGFP with C-terminal sequon fusions for DQNAT and AQNAT).Glycosylation is confirmed by an anti-6×His western blot against thetarget glycoprotein, and checked for cross-reactivity with ananti-glycan serum (α-glycan).

FIG. 16 . Glycan trimming with N-acetylgalactosaminidase enzyme onpolypeptide substrates allows for further customization of targetglycoproteins bearing engineered bacterial glycans. (A) In-gelflorescence of a FITC-labeled polypeptide substrate bearing a DQNATsequon under various glycosylation and trimming reaction conditions.Left-hand panel shows mobility shifts corresponding with full glycans,and right-hand panel shows the resulting mobility shifts for trimmedpolypeptides. X-labels show the glycans used for in-vitro glycosylation,with dilution factors in parenthesis if appropriate. (B) Glycanstructures pre- and post-trimming with N-acetylgalactosaminidase, where(a) and (c) correspond with full glycans, and (b) and (d) correspondwith trimmed structures.

FIG. 17 . Analysis of pAzF-incorporation in a cell-free glycoproteinsynthesis (CFGpS) system. (A) Echo optimization (ACH). Background levelsof incorporation in a CFGpS system were measured by adding increasingamounts of pAzF and pAzF-FS. (B) Scaled-up CFGpS and purification. CFGpSreactions were performed at two different concentrations of aaRS.Purification yields were 13.6 μM and 15.4 μM from 200 μl reactionvolumes.

FIG. 18 . Cell-free glycoprotein synthesis (CFGpS) combined withstrain-promoted alkyne-azide cycloadditions (SPAAC) to conjugate afluorophore to pAzF incorporated into a synthesized amino acid polymer.(A) Schematic of CFGpS and click chemistry using a TAMRA-labeleddibenzocyclooctyne compound. (B) and (C) SDS-PAGE analysis of purified,clicked products indicates full conversion of pAzF-incorporated acceptorprotein. (B) Coomasie Bright Blue stained (total protein). (C) In-gelfluorescence (detection of fluor).

DETAILED DESCRIPTION

Described herein are genomically recoded and engineered organisms,platforms for preparing sequence defined biopolymers in vitro comprisinga cellular extract from genomically recoded and engineered organisms,and methods for preparing sequence defined biopolymers in vitro. Theorganisms and platforms described herein allow for multi-site nsAAincorporation into sequence defined biopolymers prepared in vitro athigh yield and purity and post-incorporation modification of theincorporated nsAA in vitro, for example, via glycosylation. The use of acellular extract from the organisms results in a surprisingly high yieldfor glycoprotein production. Moreover, the use of the cellular extractresulted in surprisingly high quantities of modified glycoproteinsincorporating one or more glycosylated nsAAs. Extracts produced fromthese genomically modified organisms show surprising promise forproduction of new-kinds of sequence defined biopolymers, proteins, andin particular, glycoproteins.

The presently disclosed subject matter is described herein using severaldefinitions, as set forth below and throughout the application.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a component” should beinterpreted to mean “one or more components.”

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus ≤10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

Ranges recited herein include the defined boundary numerical values aswell as sub-ranges encompassing any non-recited numerical values withinthe recited range. For example, a range from about 0.01 mM to about 10.0mM includes both 0.01 mM and 10.0 mM. Non-recited numerical valueswithin this exemplary recited range also contemplated include, forexample, 0.05 mM, 0.10 mM, 0.20 mM, 0.51 mM, 1.0 mM, 1.75 mM, 2.5 mM 5.0mM, 6.0 mM, 7.5 mM, 8.0 mM, 9.0 mM, and 9.9 mM, among others. Exemplarysub-ranges within this exemplary range include from about 0.01 mM toabout 5.0 mM; from about 0.1 mM to about 2.5 mM; and from about 2.0 mMto about 6.0 mM, among others.

The term “amplification reaction” refers to any chemical reaction,including an enzymatic reaction, which results in increased copies of atemplate nucleic acid sequence or results in transcription of a templatenucleic acid. Amplification reactions include reverse transcription, thepolymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), and the ligase chain reaction(LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary“amplification reactions conditions” or “amplification conditions”typically comprise either two or three step cycles. Two-step cycles havea high temperature denaturation step followed by ahybridization/elongation (or ligation) step. Three step cycles comprisea denaturation step followed by a hybridization step followed by aseparate elongation step.

The terms “target, “target sequence”, “target region”, and “targetnucleic acid,” as used herein, are synonymous and refer to a region orsequence of a nucleic acid which is to be amplified, sequenced, ordetected.

The terms “nucleic acid” and “oligonucleotide,” as used herein, refer topolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide that is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide” and “polynucleotide”, and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA. For use in the presentinvention, an oligonucleotide also can comprise nucleotide analogs inwhich the base, sugar, or phosphate backbone is modified as well asnon-purine or non-pyrimidine nucleotide analogs.

Oligonucleotides can be prepared by any suitable method, includingdirect chemical synthesis by a method such as the phosphotriester methodof Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiestermethod of Brown et al., 1979, Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al., 1981, TetrahedronLetters 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066, each incorporated herein by reference. A review of synthesismethods of conjugates of oligonucleotides and modified nucleotides isprovided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187,incorporated herein by reference.

The term “hybridization,” as used herein, refers to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fullycomplementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which hybridization of fully complementarynucleic acid strands is strongly preferred are referred to as “stringenthybridization conditions” or “sequence-specific hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions; thedegree of mismatch tolerated can be controlled by suitable adjustment ofthe hybridization conditions. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically considering anumber of variables including, for example, the length and base paircomposition of the oligonucleotides, ionic strength, and incidence ofmismatched base pairs, following the guidance provided by the art (see,e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991,Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzyet al., 2008, Biochemistry, 47: 5336-5353, which are incorporated hereinby reference).

The term “primer,” as used herein, refers to an oligonucleotide capableof acting as a point of initiation of DNA synthesis under suitableconditions. Such conditions include those in which synthesis of a primerextension product complementary to a nucleic acid strand is induced inthe presence of four different nucleoside triphosphates and an agent forextension (for example, a DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature.

A primer is preferably a single-stranded DNA. The appropriate length ofa primer depends on the intended use of the primer but typically rangesfrom about 6 to about 225 nucleotides, including intermediate ranges,such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25to 150 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatenucleic acid, but must be sufficiently complementary to hybridize withthe template. The design of suitable primers for the amplification of agiven target sequence is well known in the art and described in theliterature cited herein.

Primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, that of acting as a point of initiation of DNAsynthesis. For example, primers may contain an additional nucleic acidsequence at the 5′ end which does not hybridize to the target nucleicacid, but which facilitates cloning or detection of the amplifiedproduct, or which enables transcription of RNA (for example, byinclusion of a promoter) or translation of protein (for example, byinclusion of a 5′-UTR, such as an Internal Ribosome Entry Site (IRES) ora 3′-UTR element, such as a poly(A)_(n) sequence, where n is in therange from about 20 to about 200). The region of the primer that issufficiently complementary to the template to hybridize is referred toherein as the hybridizing region.

As used herein, a primer is “specific,” for a target sequence if, whenused in an amplification reaction under sufficiently stringentconditions, the primer hybridizes primarily to the target nucleic acid.Typically, a primer is specific for a target sequence if theprimer-target duplex stability is greater than the stability of a duplexformed between the primer and any other sequence found in the sample.One of skill in the art will recognize that various factors, such assalt conditions as well as base composition of the primer and thelocation of the mismatches, will affect the specificity of the primer,and that routine experimental confirmation of the primer specificitywill be needed in many cases. Hybridization conditions can be chosenunder which the primer can form stable duplexes only with a targetsequence. Thus, the use of target-specific primers under suitablystringent amplification conditions enables the selective amplificationof those target sequences that contain the target primer binding sites.

As used herein, a “polymerase” refers to an enzyme that catalyzes thepolymerization of nucleotides. “DNA polymerase” catalyzes thepolymerization of deoxyribonucleotides. Known DNA polymerases include,for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNApolymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNApolymerase, among others. “RNA polymerase” catalyzes the polymerizationof ribonucleotides. The foregoing examples of DNA polymerases are alsoknown as DNA-dependent DNA polymerases. RNA-dependent DNA polymerasesalso fall within the scope of DNA polymerases. Reverse transcriptase,which includes viral polymerases encoded by retroviruses, is an exampleof an RNA-dependent DNA polymerase. Known examples of RNA polymerase(“RNAP”) include, for example, bacteriophage polymerases such as, butnot limited to, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymeraseand E. coli RNA polymerase, among others. The foregoing examples of RNApolymerases are also known as DNA-dependent RNA polymerase. Thepolymerase activity of any of the above enzymes can be determined bymeans well known in the art.

The term “promoter” refers to a cis-acting DNA sequence that directs RNApolymerase and other trans-acting transcription factors to initiate RNAtranscription from the DNA template that includes the cis-acting DNAsequence.

As used herein, the term “sequence defined biopolymer” refers to abiopolymer having a specific primary sequence. A sequence definedbiopolymer can be equivalent to a genetically-encoded defined biopolymerin cases where a gene encodes the biopolymer having a specific primarysequence.

As used herein, “expression template” refers to a nucleic acid thatserves as substrate for transcribing at least one RNA that can betranslated into a sequence defined biopolymer (e.g., a polypeptide orprotein). Expression templates include nucleic acids composed of DNA orRNA. Suitable sources of DNA for use a nucleic acid for an expressiontemplate include genomic DNA, cDNA and RNA that can be converted intocDNA. Genomic DNA, cDNA and RNA can be from any biological source, suchas a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecalsample, a urine sample, a scraping, among others. The genomic DNA, cDNAand RNA can be from host cell or virus origins and from any species,including extant and extinct organisms. As used herein, “expressiontemplate” and “transcription template” have the same meaning and areused interchangeably.

As used herein, “translation template” refers to an RNA product oftranscription from an expression template that can be used by ribosomesto synthesize polypeptide or protein.

As used herein, coupled transcription/translation (“Tx/Tl”), refers tothe de novo synthesis of both RNA and a sequence defined biopolymer fromthe same extract. For example, coupled transcription/translation of agiven sequence defined biopolymer can arise in an extract containing anexpression template and a polymerase capable of generating a translationtemplate from the expression template. Coupled transcription/translationcan occur using a cognate expression template and polymerase from theorganism used to prepare the extract. Coupled transcription/translationcan also occur using exogenously-supplied expression template andpolymerase from an orthogonal host organism different from the organismused to prepare the extract. In the case of an extract prepared from ayeast organism, an example of an exogenously-supplied expressiontemplate includes a translational open reading frame operably coupled abacteriophage polymerase-specific promoter and an example of thepolymerase from an orthogonal host organism includes the correspondingbacteriophage polymerase.

The term “reaction mixture,” as used herein, refers to a solutioncontaining reagents necessary to carry out a given reaction. An“amplification reaction mixture”, which refers to a solution containingreagents necessary to carry out an amplification reaction, typicallycontains oligonucleotide primers and a DNA polymerase in a suitablebuffer. A “PCR reaction mixture” typically contains oligonucleotideprimers, a DNA polymerase (most typically a thermostable DNApolymerase), dNTPs, and a divalent metal cation in a suitable buffer.

Cell-Free Protein Synthesis (CFPS) and Cell-Free Glycoprotein Synthesis(CFGpS)

The disclosed subject matter relates in part to methods, systems,components, and compositions for cell-free protein synthesis. Cell-freeprotein synthesis (CFPS) is known and has been described in the art.(See, e.g., U.S. Pat. Nos. 6,548,276; 7,186,525; 8,734,856; 7,235,382;7,273,615; 7,008,651; 6,994,986 7,312,049; 7,776,535; 7,817,794;8,298,759; 8,715,958; 9,005,920; U.S. Publication No. 2014/0349353, U.S.Publication No. 2016/0060301, U.S. Publication No. 2018/0016612, andU.S. Publication No. 2018/0016614, the contents of which areincorporated herein by reference in their entireties). A “CFPS reactionmixture” typically contains a crude or partially-purified yeast extract,an RNA translation template, and a suitable reaction buffer forpromoting cell-free protein synthesis from the RNA translation template.In some aspects, the CFPS reaction mixture can include exogenous RNAtranslation template. In other aspects, the CFPS reaction mixture caninclude a DNA expression template encoding an open reading frameoperably linked to a promoter element for a DNA-dependent RNApolymerase. In these other aspects, the CFPS reaction mixture can alsoinclude a DNA-dependent RNA polymerase to direct transcription of an RNAtranslation template encoding the open reading frame. In these otheraspects, additional NTP's and divalent cation cofactor can be includedin the CFPS reaction mixture. A reaction mixture is referred to ascomplete if it contains all reagents necessary to enable the reaction,and incomplete if it contains only a subset of the necessary reagents.It will be understood by one of ordinary skill in the art that reactioncomponents are routinely stored as separate solutions, each containing asubset of the total components, for reasons of convenience, storagestability, or to allow for application-dependent adjustment of thecomponent concentrations, and that reaction components are combinedprior to the reaction to create a complete reaction mixture.Furthermore, it will be understood by one of ordinary skill in the artthat reaction components are packaged separately for commercializationand that useful commercial kits may contain any subset of the reactioncomponents of the invention.

The strains and systems disclosed herein may be applied to cell-freeprotein methods in order to prepare glycosylated macromolecules (e.g.,glycosylated peptides, glycosylated proteins, and glycosylated lipids).Glycosylated proteins that may be prepared using the disclosed strainsand systems may include proteins having N-linked glycosylation (i.e.,glycans attached to nitrogen of asparagine and/or arginine side-chains)and/or O-linked glycosylation (i.e., glycans attached to the hydroxyloxygen of serine, threonine, tyrosine, hydroxylysine, and/orhydroxyproline). Glycosylated lipids may include O-linked glycans via anoxygen atom, such as ceramide.

The glycosylated macromolecules disclosed herein may include unbranchedand/or branched sugar chains composed of monomers as known in the artsuch as, but not limited to, glucose (e.g., β-D-glucose), galactose(e.g., β-D-galactose), mannose (e.g., β-D-mannose), fucose (e.g.,α-L-fucose), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine(GalNAc), neuraminic acid, N-acetylneuraminic acid (i.e., sialic acid),and xylose, which may be attached to the glycosylated macromolecule,growing glycan chain, or donor molecule (e.g., a donor lipid and/or adonor nucleotide) via respective glycosyltransferases (e.g.,oligosaccharyltransferases, GlcNAc transferases, GalNAc transferases,galactosyltransferases, and sialyltransferases). The glycosylatedmacromolecules disclosed herein may include glycans as known in the art.

The disclosed cell-free protein synthesis systems may utilize componentsthat are crude and/or that are at least partially isolated and/orpurified. As used herein, the term “crude” may mean components obtainedby disrupting and lysing cells and, at best, minimally purifying thecrude components from the disrupted and lysed cells, for example bycentrifuging the disrupted and lysed cells and collecting the crudecomponents from the supernatant and/or pellet after centrifugation. Theterm “isolated or purified” refers to components that are removed fromtheir natural environment, and are at least 60% free, preferably atleast 75% free, and more preferably at least 90% free, even morepreferably at least 95% free from other components with which they arenaturally associated.

Genomically Recoded Organisms

An aspect of the present invention is a genomically recoded organism(GRO) comprising a strain deficient in release factor 1 (RF1) or agenetic homolog thereof, wherein the GRO further has been engineered toexpress a heterologous RNA polymerase. GRO comprising a strain deficientin RF1 or a genetic homolog thereof may be prepared by any method ofstrain engineering. In certain embodiments the strain deficient in RF1is prepared in which all instances of the UAG codon have been removed,permitting the deletion of release factor 1 (RF1; terminates translationat UAG and UAA) and, hence, eliminating translational termination at UAGcodons. This GRO allows for the reintroduction of UAG codons, along withorthogonal translation machinery to permit efficient and site-specificincorporation of NSAAs into proteins. That is, UAG may be transformedfrom a nonsense codon (terminates translation) to a sense codon(incorporates amino acid of choice), provided the appropriatetranslation machinery is present.

The strain may comprise a prokaryote strain. In some embodiments, thestrain is an E. coli strain. In certain specific embodiments, the strainis E. coli strain C321.ΔprfA, E. coli strain rec13.ΔprfA, or aderivative of either E. coli strain C321.ΔprfA or E. coli strainrec13.ΔprfA. Other suitable strains are disclosed in U.S. PublishedApplication No. 2016/0060301, the content of which is incorporatedherein by reference in its entirety.

GROs Engineered to Express a Heterologous RNA Polymerase

In another aspect of the invention, the GRO comprising a straindeficient in RF1 or a genetic homolog thereof further comprises anadditional engineered modification in that the GRO is engineered toexpress a heterologous RNA polymerase. Suitable RNA polymerases mayinclude, but are not limited to, bacteriophage RNA polymerases.

Suitable bacteriophage RNA polymerases for the disclosed methods,systems, components and compositions may include the bacteriophage T7RNA polymerase or variants thereof. The amino acid sequence of T7 RNApolymerase is provided herein as SEQ ID NO:1 and a DNA sequence encodingT7 RNA polymerase is provided as SEQ ID NO:2. The promoter sequence forT7 RNA polymerase is provided as SEQ ID NO:3 (which may be present on atranscription template for expressing a target protein in the cell-freeprotein synthesis systems disclosed herein). In some embodiments of thedisclosed methods, systems, components and compositions, variants of T7RNA polymerase may include polymerases having at least about 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identityto SEQ ID NO:1 and/or polymerases encoded by a DNA having at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequenceidentity to SEQ ID NO:2. In some embodiments, variants of T7 RNApolymerase are resistant to cleavage by a host protease of arecombinantly engineered source strain in which the variant T7 RNApolymerase is expressed. For example, a variant of T7 RNA polymerase mayinclude a deletion of 1 or more amino acids, an insertion of one or moreamino acids, and/or one or more amino acid substitutions that make thevariant resistant to cleavage by a host protease of a recombinantlyengineered source strain in which the variant T7 RNA polymerase isexpressed. Amino acid substitutions may include replacing one or morebasic amino acids (e.g., K172, R173, K179, and/or K180) with an aminoacid that is not basic (e.g., a replacement amino acid for K172, R173,K179, and/or K180 selected from A, G, I, and L).

Suitable bacteriophage RNA polymerases for the disclosed methods,systems, components and compositions may include the bacteriophage T3RNA polymerase or variants thereof. The amino acid sequence of T3 RNApolymerase is provided herein as SEQ ID NO:4 and a DNA sequence encodingT3 RNA polymerase is provided as SEQ ID NO:5. The promoter sequence forT3 RNA polymerase is provided as SEQ ID NO:6 (which may be present on atranscription template for expressing a target protein in the cell-freeprotein synthesis systems disclosed herein). In some embodiments of thedisclosed methods, systems, components and compositions, variants of T3RNA polymerase may include polymerases having at least about 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identityto SEQ ID NO:4 and/or polymerases encoded by a DNA having at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequenceidentity to SEQ ID NO:5. In some embodiments, variants of T3 RNApolymerase are resistant to cleavage by a host protease of arecombinantly engineered source strain in which the variant T3 RNApolymerase is expressed. For example, a variant of T3 RNA polymerase mayinclude a deletion of 1 or more amino acids, an insertion of one or moreamino acids, and/or one or more amino acid substitutions that make thevariant resistant to cleavage by a host protease of a recombinantlyengineered source strain in which the variant T3 RNA polymerase isexpressed. Amino acid substitutions may include replacing one or morebasic amino acids (e.g., K173, R174, K180, and/or K181) with an aminoacid that is not basic (e.g., a replacement amino acid for K173, R174,K180, and/or K181 selected from A, G, I, and L).

Suitable bacteriophage RNA polymerases for the disclosed methods,systems, components and compositions may include the bacteriophage SP6RNA polymerase or variants thereof. The amino acid sequence of SP6 RNApolymerase is provided herein as SEQ ID NO:7 and a DNA sequence encodingSP6 RNA polymerase is provided as SEQ ID NO:8. The promoter sequence forSP6 RNA polymerase is provided as SEQ ID NO:9 (which may be present on atranscription template for expressing a target protein in the cell-freeprotein synthesis systems disclosed herein). In some embodiments of thedisclosed methods, systems, components and compositions, variants of SP6RNA polymerase may include polymerases having at least about 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identityto SEQ ID NO:7 and/or polymerases encoded by a DNA having at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide sequenceidentity to SEQ ID NO:8. In some embodiments, variants of SP6 RNApolymerase are resistant to cleavage by a host protease of arecombinantly engineered source strain in which the variant SP6 RNApolymerase is expressed. For example, a variant of SP6 RNA polymerasemay include a deletion of 1 or more amino acids, an insertion of one ormore amino acids, and/or one or more amino acid substitutions that makethe variant resistant to cleavage by a host protease of a recombinantlyengineered source strain in which the variant SP6 RNA polymerase isexpressed. Amino acid substitutions may include replacing one or more ofbasic amino acids with an amino acid that is not basic (e.g., areplacement amino acid for a basic amino acid selected from A, G, I, andL).

The GRO may be modified to express the heterologous RNA polymerase bymethods known in the art including recombination methods known in theart and as disclosed herein. In some embodiments, the GRO is modified toexpress the heterologous RNA polymerase by recombining a cassette thatexpresses the heterologous RNA polymerase into the genome of the GRO,the cassette including the coding sequence for the heterologous RNApolymerase (e.g., any of SEQ ID NOs:2, 5, 8 or a variant thereof)operably linked to a suitable promoter for expressing the RNApolymerase. Suitable promoters may include inducible promoters orconstitutive promoters. Preferably, the promoter is characterized as a“strong” promoter as described herein.

Additional GRO Modifications Including Knock-Out Mutations

Optionally, the GRO comprising a strain deficient in RF1 or a genetichomolog thereof and engineered to express a heterologous RNA polymerasefurther comprises at least one additional genetic knock-out mutation.The at least one additional genetic knock-out mutation is preferably aknock-out mutation that downregulates or eliminates a negative proteineffector for CFPS. In certain embodiments, the at least one additionalgenetic knock-out mutation improves DNA stability, RNA stability,protein stability, amino acid stability, energy supply, or anycombination thereof. In certain embodiments, the at least one additionalgenetic knock-out mutation comprises 1, 2, 3, 4, or more than 4 geneticknock-out mutations. In embodiments where the strain comprises 2 or moregenetic knock-out mutations, at least 2 of the genetic knock-outmutations may both improve the same attribute, improved DNA stability,improved RNA stability, improved protein stability, improved amino acidstability, improved energy supply, or may both improve differentattributes.

To improve DNA or RNA stability, the at least one additional geneticknock-out mutation may target the functional inactivation of nucleases.In vivo, nucleases play important roles in regulating DNA and mRNAturnover. However, their presence in crude cell extracts is expected tobe deleterious, leading to template instability and reactiontermination. A nonexhaustive list of potential negative effectorsfollow: RNase A (encoded by ma) degrades RNA by catalyzing the cleavageof phosodiester bonds, and identification of strains (e.g., MRE600, A19)lacking ma was important for early studies in in vitro translation.RNase II (encoded by rnb) is responsible for mRNA decay by 3′ to 5′exonuclease activity, and cell extracts lacking RNase II exhibit a 70%increase in CFPS efficiency. RNase E (encoded by me) is part of a coldshock degradosome that induces mRNA decay in cold shock, which the cellsexperience during harvest prior to extract generation. MazF (encoded bymazF) is a toxin that degrades mRNA by sequence-specific (ACA)endoribonuclease activity, which could affect transcript stability. CsdA(encoded by csdA) is part of a cold shock degradosome along with RNase Eand induces mRNA decay in cold shock, which the cells experience duringharvest prior to extract generation. DNA-specific endonuclease I(encoded by endA) breaks double-stranded DNA, and its deletion haspreviously shown to be important for extending the duration of CFPSreactions. These and other nucleases may be functionally inactivated bythe at least on additional genetic knock-out mutation.

To improve protein stability, the at least one additional geneticknock-out mutation may target the functional inactivation of proteases.In vivo, these proteases play important roles in regulating proteinturnover. However, their presence in CFPS reactions is expected to bedeleterious, leading to protein instability issues. A nonexhaustive listof potential negative effectors follow: Glutathione reductase (encodedby gor) reduces oxidized glutathione to maintain a reducing environmentin the cytoplasm of a cell, making synthesis of disulfide-bondedproteins problematic. Lon (encoded by ion) is an ATP-dependent proteasethat demonstrated improved protein production in cell-free systems inBL21 strains upon transcriptional down regulation. Outer membraneprotease VII (encoded by ompT) demonstrates specificity for paired basicresidues and has been shown to stabilize proteins during CFPS uponremoval. These and other proteases may be functionally inactivated bythe at least on additional genetic knock-out mutation.

The at least one additional genetic knock-out mutation may targetproteins known to negatively affect amino acid or energy supply. Invivo, these proteins play important roles in metabolism and substrateturnover. However, their presence in crude cell extracts is expected tobe deleterious, leading to decreased amino acid and energy supply tosupport translation. A nonexhaustive list of potential negativeeffectors follow: Glutamate dehydrogenase (encoded by gdhA) catalyzesthe deamination of glutamate, which may affect glutamate's stability.Glutamate-cysteine-ligase (encoded by gshA) catalyzes the first step ofglutathione synthesis and may decrease the stability of cysteine. Serinedeaminase I (encoded by sdaA) and serine deaminase II (encoded by sdaB)are two of the three enzymes involved in serine degradation. Argininedecarboxylase (encoded by speA) consumes arginine in the biosyntheticproduction of putrescine. Tryptophanase (encoded by tnaA) consumestryptophan in the production of indole. Lastly, glycerol kinase (encodedby glpK) consumes ATP to phosphorylate glycerol, which could helpdeplete the energy supply required for cell-free reactions. These andother proteins may be functionally inactivated by the at least onadditional genetic knock-out mutation.

In some embodiments, the disclosed strains may include a genomicmodification that results in a deficiency in expression of endA and orgor, in particular. In some embodiments, the disclosed strains mayinclude a genomic modification that results in a deletion of at least aportion of endA and or gor, in particular.

In some embodiments, the disclosed strains may include a genomicmodification which inactivates and/or deletes a gene selected from gmd,waal or both of gmd and waal, or genetic equivalents thereof. When themodified strain is E. coli, the modification may include an inactivatingmodification in the gmd gene (e.g., via a deletion of at least a portionof the gmd gene). The sequence of the E. coli gmd gene is providedherein as SEQ ID NO:1 and the amino acid sequence of E. coli GDP-mannose4,6-dehydratase is provided as SEQ ID NO:2. When the modified strain isE. coli, the modification may include an inactivating modification inthe waal gene (e.g., via a deletion of at least a portion of the waalgene). The sequence of the E. coli waaL gene is provided herein as SEQID NO:3 and the amino acid sequence of E. coli O-antigen ligase isprovided as SEQ ID NO:4.

Strains having at least one additional genetic knock-out mutation may beprepared by any method of engineering a strain to functionallyinactivate the negative effector to lessen or eliminate the negativeeffector from a lysate prepared from the strain. In certain embodiments,the genetic knock-out mutations may be prepared by inserting either anonsense mutation and/or a frameshift mutation into the genome of thestrain as well as deleting a vital portion of a gene coding sequence. Incertain embodiments, the genetic knock-out mutations may be prepared byremoving regulatory sequences (i.e. promoter, ribosome binding site) orotherwise changing these sequences in the genome as to render themnon-functional. In certain embodiments, negative effectors can befunctionally knocked out in lysates by introducing a unique affinity tagand subsequently using the tag to selectively remove the effectorprotein from the lysates. In certain embodiments a strain having atleast one additional genetic knock-out mutation may be prepared bymultiplex automated genome engineering (MAGE), λ-Redrecombinase-mediated recombination (Datsenko-Wanner), zinc-fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein-9 nuclease (Cas9), and any other commonlyused recombineering and genome engineering tools.

Additional GRO Modifications Including Upregulated Gene Products

Optionally, the GRO comprising a strain deficient in RF1 or a genetichomolog thereof further and engineered to express a heterologous RNApolymerase further comprises at least one additional upregulated geneproduct. In certain embodiments the GRO comprising a strain deficient inRF1 or a genetic homolog thereof further comprises at least oneadditional upregulated gene product and at least one additional geneticknock-out mutation. The at least one additional upregulated gene productis preferably an upregulated gene product that is a positive effectorfor CFPS. In certain embodiments, the at least one additionalupregulated gene product improves energy supply, chaperone levels,translations function, ribosome recycling, or any combination thereof.In certain embodiments, the at least on additional upregulated geneproduct comprises 1, 2, 3, 4, or more than 4 upregulated gene products.In embodiments where the strain comprises 2 or more upregulated geneproducts, at least 2 of the upregulated gene products may both improvethe same attribute, improved energy supply, improved chaperone levels,improved translation function, or improved ribosome recycling, or mayboth improve different attributes.

To improve energy supply, the at least one additional upregulated geneproduct may target the upregulation of kinases. In vivo, these proteinsplay important roles in metabolism and the transfer of phosphate groups.The upregulated presence in crude cell extracts is expected to improveenergy supply to support translation. A nonexhaustive list of potentialpositive effectors follow: Acetate kinase (encoded by ackA) increasesthe overall metabolic flux of metabolites toward substrate-level ATPgeneration. Nucleoside-diphosphate kinase (encoded by ndk) facilitatethe synthesis of NTPs from their corresponding NDPs. Pyruvate kinasemonomer (encoded by pykF) helps drive ATP generation. These and otherkinases may be the at least one additional upregulated gene product.

To improve energy supply, the at least one additional upregulated geneproduct may target the upregulate of deaminases. In vivo, these proteinsmay play important roles in metabolism and preparing metabolites. Anonexhaustive list of potential positive effectors follow: Cytidinedeaminase (encoded by cdd) initiates the deamination of cytidine whichmay lead to the synthesis of UTP. These and other deaminases may be theat least one additional upregulated gene product

To improve chaperone levels, the at least one upregulated gene productmay target the upregulation of isomerases, foldases and/or holdases. Invivo, these proteins may play important roles in the assisting proteinsto adopt functionally active conformations. The upregulated presence incrude cell extracts is expected to improve chaperone levels to supportprotein production into soluble and/or active confirmations. Anonexhaustive list of potential positive effectors follow: Disulfidebond isomerase (encoded by dsbC) shuffles disulfide bonds into correctpositions. Chaperone protein DnaK (encoded by dnaK) aids the folding ofnascent polypeptide chains and the rescue of misfolded proteins.Chaperone protein DnaJ (encoded by danJ) stimulates the ATPase activityof DnaK. Protein GrpE (encoded by grpE) stimulates the ATPas activity ofDnaK. Trigger Factor (encoded by tig) aids the folding of nascentpolypeptides. The 10 kDa chaperonin subunit (encoded by groS) forms partof the GroEL-GroES chaperonin complex that aids in protein folding. The60 kDa chaperonin subunit (encoded by groL) forms part of theGroEL-GroES chaperonin complex that aids in protein folding. These andother isomerases, foldases, and/or holdases may be the at least oneadditional upregulated gene product.

To improve translation function, the at least one upregulated geneproduct may target the upregulation of initiation factors and/orelongation factors. In vivo, these proteins play important roles in thetranslation function. The upregulated presence in crude cell extracts isexpected to improve translation function. A nonexhaustive list ofpotential positive effectors follow: Translation initiation factor IF-1(encoded by infA) interacts with the 30S ribosomal subunit to initiatetranslations. Translation initiation faction IF-2 (encoded by infB) hasa role in the proper placement of the charged initiator fMet-tRNA via aGTP-dependent mechanism. Elongations factor G (encoded by fusA)facilitates translocation of the ribosome by one codon along an mRNA.Elongation factor P (encoded by efp) stimulates the synthesis of peptidebonds. Elongation factor 4 (encoded by lepA) can alter the rate oftranslation, leading to increases in the rate of translation undercertain stress conditions. Elongation factor TU 2 (encoded by tufB)helps shuttle charge tRNAs to ribosomes. These and other initiationfactors and/or elongation factors may be the at least one additionalupregulated gene product.

To improve translation function, the at least one upregulated geneproduct may target the upregulation of recycling factors. In vivo, theseproteins play important roles in the ribosome recycling. The upregulatedpresence in crude cell extracts is expected to improve ribosomerecycling. A nonexhaustive list of potential positive effectors follow:Heat shock protein 15 (encoded by hslR) is involved with the recyclingof free 50S ribosomal subunits. Ribosome-recycling factor (encoded byfrr) promotes rapid recycling of ribosomal subunits after the release ofthe polypeptide chain. These and other recycling factors may be the atleast one additional upregulated gene product.

Strains having at least one additional genetic knock-out mutation, maybe prepared by any method of engineering a strain to functionallyincrease a positive effector to increase the presence of the positiveeffector in the lysate prepared from the strain. In certain embodiments,the upregulated gene product is expressed from a plasmid present in theGRO and/or expressed from an integration site in GRO genome.Additionally, gene upregulation may be enhanced by engineering thepromoter and/or ribosome binding site in front of your gene of interestlocated either on a plasmid or on the genome. A strongerpromoter/ribosome binding site would lead to an increase intranscriptional activity. Techniques commonly employed to integrate aplasmid overexpressing a positive effector into a strain includestransformation. Techniques commonly employed to integrate a genecassette containing a positive effector into the genome foroverexpression includes X-Red recombinase-mediated recombination(Datsenko-Wanner).

Platforms for Preparing Sequence Defined Biopolymers

An aspect of the invention is a platform for preparing a sequencedefined biopolymer of protein in vitro. The platform for preparing asequence defined polymer or protein in vitro comprises a cellularextract from the GRO organism as described above. Because CFPS exploitsan ensemble of catalytic proteins prepared from the crude lysate ofcells, the cell extract (whose composition is sensitive to growth media,lysis method, and processing conditions) is the most critical componentof extract-based CFPS reactions. A variety of methods exist forpreparing an extract competent for cell-free protein synthesis,including U.S. patent application Ser. No. 14/213,390 to Michael C.Jewett et al., entitled METHODS FOR CELL-FREE PROTEIN SYNTHESIS, filedMar. 14, 2014, and now published as U.S. Patent Application PublicationNo. 2014/0295492 on Oct. 2, 2014, and U.S. patent application Ser. No.14/840,249 to Michael C. Jewett et al., entitled METHODS FOR IMPROVED INVITRO PROTEIN SYNTHESIS WITH PROTEINS CONTAINING NON STANDARD AMINOACIDS, filed Aug. 31, 2015, and now published as U.S. Patent ApplicationPublication No. 2016/0060301, on Mar. 3, 2016, the contents of which areincorporated by reference.

The platform may comprise an expression template, a translationtemplate, or both an expression template and a translation template. Theexpression template serves as a substrate for transcribing at least oneRNA that can be translated into a sequence defined biopolymer (e.g., apolypeptide or protein). The translation template is an RNA product thatcan be used by ribosomes to synthesize the sequence defined biopolymer.In certain embodiments the platform comprises both the expressiontemplate and the translation template. In certain specific embodiments,the platform may be a coupled transcription/translation (“Tx/Tl”) systemwhere synthesis of translation template and a sequence definedbiopolymer from the same cellular extract.

The platform may comprise one or more polymerases capable of generatinga translation template from an expression template. The polymerase maybe supplied exogenously or may be supplied from the organism used toprepare the extract. In certain specific embodiments, the polymerase isexpressed from a plasmid present in the organism used to prepare theextract and/or an integration site in the genome of the organism used toprepare the extract.

The platform may comprise an orthogonal translation system. Anorthogonal translation system may comprise one or more orthogonalcomponents that are designed to operate parallel to and/or independentof the organism's orthogonal translation machinery. In certainembodiments, the orthogonal translation system and/or orthogonalcomponents are configured to incorporation of unnatural amino acids. Anorthogonal component may be an orthogonal protein or an orthogonal RNA.In certain embodiments, an orthogonal protein may be an orthogonalsynthetase. In certain embodiments, the orthogonal RNA may be anorthogonal tRNA or an orthogonal rRNA. An example of an orthogonal rRNAcomponent has been described in Application No. PCT/US2015/033221 toMichael C. Jewett et al., entitled TETHERED RIBOSOMES AND METHODS OFMAKING AND USING THEREOF, filed 29 May 2015, and now published asWO2015184283, and U.S. patent application Ser. No. 15/363,828, toMichael C. Jewett et al., entitled RIBOSOMES WITH TETHERED SUBUNITS,filed on Nov. 29, 2016, and now published as U.S. Patent ApplicationPublication No. 2017/0073381, on Mar. 16, 2017, the contents of whichare incorporated by reference. In certain embodiments, one or moreorthogonal components may be prepared in vivo or in vitro by theexpression of an oligonucleotide template. The one or more orthogonalcomponents may be expressed from a plasmid present in the genomicallyrecoded organism, expressed from an integration site in the genome ofthe genetically recoded organism, co-expressed from both a plasmidpresent in the genomically recoded organism and an integration site inthe genome of the genetically recoded organism, express in the in vitrotranscription and translation reaction, or added exogenously as a factor(e.g., a orthogonal tRNA or an orthogonal synthetase added to theplatform or a reaction mixture).

Altering the physicochemical environment of the CFPS reaction to bettermimic the cytoplasm can improve protein synthesis activity. Thefollowing parameters can be considered alone or in combination with oneor more other components to improve robust CFPS reaction platforms basedupon crude cellular extracts (for examples, S12, S30 and S60 extracts).

The temperature may be any temperature suitable for CFPS. Temperaturemay be in the general range from about 10° C. to about 40° C., includingintermediate specific ranges within this general range, include fromabout 15° C. to about 35° C., form about 15° C. to about 30° C., formabout 15° C. to about 25° C. In certain aspects, the reactiontemperature can be about 15° C., about 16° C., about 17° C., about 18°C., about 19° C., about 20° C., about 21° C., about 22° C., about 23°C., about 24° C., about 25° C.

The CFPS reaction can include any organic anion suitable for CFPS. Incertain aspects, the organic anions can be glutamate, acetate, amongothers. In certain aspects, the concentration for the organic anions isindependently in the general range from about 0 mM to about 200 mM,including intermediate specific values within this general range, suchas about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM,about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,about 160 mM, about 170 mM, about 180 mM, about 190 mM and about 200 mM,among others.

The CFPS reaction can also include any halide anion suitable for CFPS.In certain aspects the halide anion can be chloride, bromide, iodide,among others. A preferred halide anion is chloride. Generally, theconcentration of halide anions, if present in the reaction, is withinthe general range from about 0 mM to about 200 mM, includingintermediate specific values within this general range, such as thosedisclosed for organic anions generally herein.

The CFPS reaction may also include any organic cation suitable for CFPS.In certain aspects, the organic cation can be a polyamine, such asspermidine or putrescine, among others. Preferably polyamines arepresent in the CFPS reaction. In certain aspects, the concentration oforganic cations in the reaction can be in the general about 0 mM toabout 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 mM. Incertain aspects, more than one organic cation can be present.

The CFPS reaction can include any inorganic cation suitable for CFPS.For example, suitable inorganic cations can include monovalent cations,such as sodium, potassium, lithium, among others; and divalent cations,such as magnesium, calcium, manganese, among others. In certain aspects,the inorganic cation is magnesium. In such aspects, the magnesiumconcentration can be within the general range from about 1 mM to about50 mM, including intermediate specific values within this general range,such as about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM,about 7 mM, about 8 mM, about 9 mM, about 10 mM, among others. Inpreferred aspects, the concentration of inorganic cations can be withinthe specific range from about 4 mM to about 9 mM and more preferably,within the range from about 5 mM to about 7 mM.

The CFPS reaction includes NTPs. In certain aspects, the reaction useATP, GTP, CTP, and UTP. In certain aspects, the concentration ofindividual NTPs is within the range from about 0.1 mM to about 2 mM.

The CFPS reaction can also include any alcohol suitable for CFPS. Incertain aspects, the alcohol may be a polyol, and more specificallyglycerol. In certain aspects the alcohol is between the general rangefrom about 0% (v/v) to about 25% (v/v), including specific intermediatevalues of about 5% (v/v), about 10% (v/v) and about 15% (v/v), and about20% (v/v), among others.

Cell-Free Glycoprotein Synthesis (CFGpS) in Prokaryotic Cell LysatesEnriched with Components for Glycosylation

The disclosed GRO's, compositions, and methods may be utilized forperforming cell-free glycoprotein synthesis (CFGpS). In someembodiments, the composition and methods include or utilize prokaryoticcell lysates enriched with components for glycosylation and preparedfrom genetically modified strains of prokaryotes.

In some embodiments, the genetically modified prokaryote is agenetically modified strain of Escherichia coli or any other prokaryotesuitable for preparing a lysate for CFGpS. Optionally, the modifiedstrain of Escherichia coli is derived from rEc.C321. Preferably, themodified strain includes genomic modifications (e.g., deletions of genesrendering the genes inoperable) that preferably result in lysatescapable of high-yielding cell-free protein synthesis. Also, preferably,the modified strain includes genomic modification (e.g., deletions ofgenes rendering the genes inoperable) that preferably result in lysatescomprising sugar precursors for glycosylation at relatively highconcentrations (e.g., in comparison to a strain not having the genomicmodification). In some embodiments, a lysate prepared from the modifiedstrain comprises sugar precursors at a concentration that is at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or higher thana lysate prepared from a strain that is not modified.

In some embodiments, the modified strain includes a modification thatresults in an increase in the concentration of a monosaccharide utilizedin glycosylation (e.g., glucose, mannose, N-acetyl-glucosamine (GlcNAc),N-acetyl-galactosamine (GalNAc), galactose, sialic acid, neuraminicacid, fucose). As such, the modification may inactivate an enzyme thatmetabolizes a monosaccharide or polysaccharide utilized inglycosylation. In some embodiments, the modification inactivates adehydratase or carbon-oxygen lyase enzyme (EC 4.2) (e.g., via a deletionof at least a portion of the gene encoding the enzyme). In particular,the modification may inactivate a GDP-mannose 4,6-dehydratase (EC4.2.1.47). When the modified strain is E. coli, the modification mayinclude an inactivating modification in the gmd gene (e.g., via adeletion of at least a portion of the gmd gene). The sequence of the E.coli gmd gene is provided herein as SEQ ID NO:1 and the amino acidsequence of E. coli GDP-mannose 4,6-dehydratase is provided as SEQ IDNO:2.

In some embodiments, the modified strain includes a modification thatinactivates an enzyme that is utilized in the glycosyltransferasepathway. In some embodiments, the modification inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). In particular, themodification may inactivate an O-antigen ligase that optionallyconjugates an O-antigen to a lipid A core oligosaccharide. Themodification may include an inactivating modification in the waaL gene(e.g., via a deletion of at least a portion of the waaL gene). Thesequence of the E. coli waaL gene is provided herein as SEQ ID NO:3 andthe amino acid sequence of E. coli O-antigen ligase is provided as SEQID NO:4.

In some embodiments, the modified strain includes a modification thatinactivates a dehydratase or carbon-oxygen lyase enzyme (e.g., via adeletion of at least a portion of the gene encoding the enzyme) and alsothe modified strain includes a modification that inactivates anoligosaccharide ligase enzyme (e.g., via a deletion of at least aportion of the gene encoding the enzyme). The modified strain mayinclude an inactivation or deletion of both gmd and waaL.

In some embodiments, the modified strain may be modified to express oneor more orthogonal or heterologous genes. In particular, the modifiedstrain may be genetically modified to express an orthogonal orheterologous gene that is associated with glycoprotein synthesis such asa glycosyltransferase (GT) which is involved in the lipid-linkedoligosaccharide (LLO) pathway. In some embodiments, the modified strainmay be modified to express an orthogonal or heterologousoligosaccharyltransferase (EC 2.4.1.119) (OST).Oligosaccharyltransferases or OSTs are enzymes that transferoligosaccharides from lipids to proteins.

In particular, the modified strain may be genetically modified toexpress an orthogonal or heterologous gene in a glycosylation system(e.g., an N-linked glycosylation system and/or an O-linked glycosylationsystem). The N-linked glycosylation system of Campylobacter jejuni hasbeen transferred to E. coli. (See Wacker et al., “N-linked glycosylationin Campylobacter jejuni and its functional transfer into E. coli,”Science 2002, Nov. 29; 298(5599):1790-3, the content of which isincorporated herein by reference in its entirety). In particular, themodified strain may be modified to express one or more genes of the pgllocus of C. jejuni or C. lari or one or more genes of a homologous pgllocus. The genes of the pgl locus include pglG, pglF, pglE, wlaJ, pglD,pglC, pglA, pglB, pglJ, pglI, pglH, pglK, and gne, and are used tosynthesize lipid-linked oligosaccharides (LLOs) and transfer theoligosaccharide moieties of the LLOs to a protein via anoligosaccharyltransferase.

Suitable orthogonal or heterologous oligosaccharyltransferases (OST)which may be expressed in the genetically modified strains may includeC. jejuni or C. lari oligosaccharyltransferase PglB. The gene for the C.jejuni OST is referred to as pg/B, which sequence is provided as SEQ IDNO:5 and the amino acid sequence of C. jejuni PglB is provided as SEQ IDNO:6. PglB catalyzes transfer of an oligosaccharide to a D/E-Y-N-X-S/Tmotif (Y, X≠P) present on a protein.

Crude cell lysates may be prepared from the modified strains disclosedherein. The crude cell lysates may be prepared from different modifiedstrains as disclosed herein and the crude cell lysates may be combinedto prepare a mixed crude cell lysate. In some embodiments, one or morecrude cell lysates may be prepared from one or more modified strainsincluding a genomic modification (e.g., deletions of genes rendering thegenes inoperable) that preferably result in lysates comprising sugarprecursors for glycosylation at relatively high concentrations (e.g., incomparison to a strain not having the genomic modification). In someembodiments, one or more crude cell lysates may be prepared from one ormore modified strains that have been modified to express one or moreorthogonal or heterologous genes or gene clusters that are associatedwith glycoprotein synthesis. Preferably, the crude cell lysates or mixedcrude cell lysates are enriched in glycosylation components, such aslipid-linked oligosaccharides (LLOs), glycosyltransferases (GTs),oligosaccharyltransferases (OSTs), or any combination thereof. Morepreferably, the crude cell lysates or mixed crude cell lysates areenriched in Man₃GlcNAc₂ LLOs representing the core eukaryotic glycanand/or Man₃GlcNAc₄Gal₂Neu₅Ac₂ LLOs representing the fully sialylatedhuman glycan.

The disclosed crude cell lysates may be used in cell-free glycoproteinsynthesis (CFGpS) systems to synthesize a variety of glycoproteins. Theglycoproteins synthesized in the CFGpS systems may include prokaryoticglycoproteins and eukaryotic proteins, including human proteins. TheCFGpS systems may be utilized in methods for synthesizing glycoproteinsin vitro by performing the following steps using the crude cell lysatesor mixtures of crude cell lysates disclosed herein: (a) performingcell-free transcription of a gene for a target glycoprotein; (b)performing cell-free translation; and (c) performing cell-freeglycosylation. The methods may be performed in a single vessel ormultiple vessels. Preferably, the steps of the synthesis method may beperformed using a single reaction vessel. The disclosed methods may beused to synthesis a variety of glycoproteins, including prokaryoticglycoproteins and eukaryotic glycoproteins.

Methods for Preparing Proteins and Sequence Defined Biopolymers

An aspect of the invention is a method for cell-free protein synthesisof a sequence defined biopolymer or protein in vitro. The methodcomprises contacting a RNA template encoding a sequence definedbiopolymer with a reaction mixture comprising a cellular extract from aGRO as described above. Methods for cell-free protein synthesis of asequence defined biopolymers have been described.

In certain embodiments, a sequence-defined biopolymer or proteincomprises a product prepared by the method or the platform that includesan amino acids. In certain embodiments the amino acid may be a naturalamino acid. As used herein a natural amino acid is a proteinogenic aminoacid encoded directly by a codon of the universal genetic code. Incertain embodiments the amino acid may be an unnatural amino acid. Asused here an unnatural amino acid is a nonproteinogenic amino acid. Anunnatural amino acids may also be referred to as a non-standard aminoacid (NSAA) or non-canonical amino acid. In certain embodiments, asequence defined biopolymer or protein may comprise a plurality ofunnatural amino acids. In certain specific embodiments, a sequencedefined biopolymer or protein may comprise a plurality of the sameunnatural amino acid. The sequence defined biopolymer or protein maycomprise at least 5, at least 10, at least 15, at least 20, at least 25,at least 30, at least 35, or at least 40 or the same or differentunnatural amino acids.

Examples of unnatural, non-canonical, and/or non-standard amino acidsinclude, but are not limited, to a p-azido-L-phenylalanine, ap-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcpβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an unnatural analogue of a methionine amino acid;an unnatural analogue of a leucine amino acid; an unnatural analogue ofa isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, 24ufa24hor, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or a combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; a metal binding amino acid; a metal-containingamino acid; a radioactive amino acid; a photocaged and/orphotoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol or polyether; a heavy atom substituted amino acid; achemically cleavable or photocleavable amino acid; an amino acid with anelongated side chain; an amino acid containing a toxic group; a sugarsubstituted amino acid; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an a-hydroxy containing acid; an amino thioacid; an α,α disubstituted amino acid; a β-amino acid; a γ-amino acid, acyclic amino acid other than proline or histidine, and an aromatic aminoacid other than phenylalanine, tyrosine or tryptophan.

The methods described herein allow for preparation of sequence definedbiopolymers or proteins with high fidelity to a RNA template. In otherwords, the methods described herein allow for the correct incorporationof unnatural, non-canonical, and/or non-standard amino acids as encodedby an RNA template. In certain embodiments, the sequence definedbiopolymer encoded by a RNA template comprises at least 5, at least 10,at least 15, at least 20, at least 25, at least 30, at least 35, or atleast 40 unnatural, non-canonical, and/or non-standard amino acids and aproduct prepared from the method includes at least 80%, at least 85%, atleast 90%, at least 95%, or 100% of the encoded unnatural,non-canonical, and/or non-standard amino acids.

The methods described herein also allow for the preparation of aplurality of products prepared by the method. In certain embodiments, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98% ofa plurality of products prepared by the method are full length. Incertain embodiments, the sequence defined biopolymer encoded by a RNAtemplate comprises at least 5, at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, or at least 40 unnatural,non-canonical, and/or non-standard amino acids and at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% of a plurality ofproducts prepared by the method include 100% of the encoded unnatural,non-canonical, and/or non-standard amino acids.

In certain embodiments, the sequence defined biopolymer or the proteinencodes a therapeutic product, a diagnostic product, a biomaterialproduct, an adhesive product, a biocomposite product, or an agriculturalproduct.

Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC)

Strain-promoted alkyne-azide cycloadditions (SPAAC) and the use of SPAACin cell-free glycoprotein synthesis are contemplated herein. SPAAC hasbeen described. (See, e.g., Mbua et al., Chembiochem. 2011 Aug. 16;12(12):1912-21; Darabedian et al., Biochemistry 2018 Oct. 9; 57 (40):5769-5774; Zhang et al., Molecules 2013, 18, 7145-7145; the contents ofwhich are incorporated herein by reference in their entireties). Thedisclosed compositions and methods may comprise and/or utilizecomponents for performing SPPAC reactions, which may include, but arenot limited components comprising dibenzocyclooctyne (DBCO) moieties andderivatives thereof such as DBCO-amine, DBCO-acid,DBCO-N-hydroxysuccinimidyl ester, DBCO-maleimide, and PEGylated formsthereof such as DBCO-PEG4-acid. In some embodiments, the componentcomprising a DBCO moiety may be conjugated to a saccharide or afluorophore (e.g., TAMRA) and the DBCO moiety may be utilized to linkthe saccharide or the fluorophore to a non-standard amino acid (e.g.,pAzF) of an amino acid polymer via click chemistry in a cell-freeglycoprotein synthesis (CFGpS) platform.

Miscellaneous

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred aspects of this invention are described herein, including thebest mode known to the inventors for carrying out the invention.Variations of those preferred aspects may become apparent to those ofordinary skill in the art upon reading the foregoing description. Theinventors expect a person having ordinary skill in the art to employsuch variations as appropriate, and the inventors intend for theinvention to be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and should not be interpretedto limit the scope of the claimed subject matter.

Embodiment 1. Escherichia coli chassis strains with genomicmodifications that result in lysates capable of high-yielding cell-freeprotein synthesis and which accumulate sugar precursors forglycosylation.

Embodiment 2. The Escherichia coli chassis strains of embodiment 1,wherein the Escherichia coli chassis strains are based on genomicallyrecoded E. coli that lack release factor 1 with genomic modificationsthat result in lysates capable of high-yielding cell-free proteinsynthesis and which accumulate sugar precursors for glycosylation.

Embodiment 3. Escherichia coli chassis strains derived from rEc.C321prfA- gor- endA- with genomic modifications that result in lysatescapable of high-yielding cell-free protein synthesis and whichaccumulate sugar precursors for glycosylation.

Embodiment 4. The strains described in any of embodiments 1-3, in whichthe genomic modification is an inactivation or deletion of gmd.

Embodiment 5. The strains described in any of embodiments 1-4, in whichthe genomic modification is an inactivation or deletion of waaL.

Embodiment 6. The strains described in any of embodiments 1-5, in whichthe genomic modification is an inactivation or deletion of both gmd andwaaL.

Embodiment 7. A method for crude cell lysate preparation in whichorthogonal genes or gene clusters are expressed in the source strain,which results in lysates enriched with glycosylation components(lipid-linked oligosaccharides (LLOs), oligosaccharyltransferases(OSTs), or both LLOs and OST) and (an) orthogonal translation system(s)(OTS) (orthogonal tRNA(s), engineered aminoacyltRNA synthetase(s)(aaRS), engineered elongation factors(s) (EF), or any combination oftRNAs, aaRSs, and elongation factors), optionally wherein the crude celllysate is prepared from any of the strains described in embodiments 1-6.

Embodiment 8. The method of embodiment 7, in which the source strainoverexpresses a glycosyltransferase pathway from C. jejuni, resulting inthe production of C. jejuni lipid-linked oligosaccharides (LLOs).

Embodiment 9. The method of embodiment 7 or 8, in which the sourcestrain overexpresses a glycosyltransferase pathway from C. lari,resulting in the production of C. lari lipid-linked oligosaccharides(LLOs).

Embodiment 10. The method of any of embodiments 7-9, in which the sourcestrain overexpresses a synthetic glycosyltransferase pathway, resultingin the production of GlcNAcGalNAc5 lipid-linked oligosaccharides (LLOs).

Embodiment 11. The method of any of embodiments 7-10, in which the OSTis a naturally occurring bacterial homolog of C. jejuni PglB.

Embodiment 12. The method of any of embodiments 7-11, in which the OSTis an engineered variant of C. jejuni PglB.

Embodiment 13. The method of any of embodiments 7-12, in which the OSTis a naturally occurring archaeal OST.

Embodiment 14. The method of any of embodiments 7-13, in which the OSTis a naturally occurring single-subunit eukaryotic OST, such as thosefound in Trypanosoma bruceii.

Embodiment 15. The method of any of embodiments 7-14, in which thesource strain overexpresses a synthetic glycosyltransferase pathway,resulting in the production of Man3GlcNAc2 lipid-linked oligosaccharides(LLOs).

Embodiment 16. The method of any of embodiments 7-15, in which thesource strain overexpresses a synthetic glycosyltransferase pathway,resulting in the production of Man5GlcNAc3 lipid-linked oligosaccharides(LLOs).

Embodiment 17. The method of any of embodiments 7-16, in which thesource strain overexpresses a glycosyltransferase pathway and an OST,resulting in the production of LLOs and OST.

Embodiment 18. The method of any of embodiments 7-16, in which thesource strain overexpresses an O antigen glycosyltransferase pathway,resulting in the production of O antigen lipid-linked oligosaccharides(LLOs).

Embodiment 19. The method of any of embodiments 7-18, in which the OTSis engineered to install p-azido-L-phenylalanine (pAzF).

Embodiment 20. The method of any of embodiments 7-19, in which the OTSis engineered to install p-propargyloxy-L-phenylalanine (pAcF).

Embodiment 21. The method of any of embodiments 7-20, in which the OTSinstalls phosphoserine (Sep).

Embodiment 22. The method of any of embodiments 7-21, in which the OTSinstalls any amino acid other than the 20 canonical amino acids.

Embodiment 23. A method for cell-free production of nsAA-containingglycoproteins that involves crude cell lysates.

Embodiment 24. The method of embodiment 23, in which the immunogeniccarrier is a protein.

Embodiment 25. The method of embodiment 23 or 24, in which theimmunogenic carrier is a peptide.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1—Coordinated Non-Standard Amino Acid Incorporation and ProteinGlycosylation in Crude Cell Lysates

Abstract

Glycosylation, or the attachment of glycans (sugars) to proteins, is themost abundant post-translational modification in nature and plays apivotal role in protein folding, sorting, and activity. In molecularmedicine, the compositions and patterns of glycans on recombinanttherapeutic glycoproteins are known to impact pharmacokinetics and drugactivity. The inability to precisely control protein glycosylation withcurrent technologies represents a key challenge in the fields ofglycoprotein synthesis and glycoprotein therapeutics. To address thischallenge, PEGylation, or the chemical conjugation of hydrophilicpoly(ethylene glycol) (PEG) polymers to proteins, has been used to mimicthe effects of glycosylation to a certain extent by improving watersolubility, increasing bioavailability, modulating immunogenicity, andextending therapeutic protein half-life in vivo. This strategy has beenespecially useful in generating long-acting forms of small proteinhormones that are naturally glycosylated, such as PEGylated humanerythropoietin (Mircera) and PEGylated human growth hormone(Somavert/Pegvisomant). However, the therapeutic efficacy andbiochemical properties of molecules that are both PEGylated andglycosylated has not vet been explored.

Here, we describe a CFGpS system with the potential to enablecontrollable glycosylation of therapeutic proteins containingbio-orthogonal chemical handles for site-specific chemical conjugationof PEG or other chemical groups, including drug conjugates (e.g.,antibody-drug conjugates or ADCs). This is accomplished through theco-translational incorporation of non-standard amino acids (nsAAs) andthe post-translational attachment of gly cans to specific amino acidsequences (sequons) in the protein of interest. Uniquely, in ourplatform technology, i) all the biosynthetic machinery for proteinsynthesis and glycosylation is supplied by the E. coli lysate and ii)transcription, translation (including non-standard amino acidincorporation), and glycosylation occur in an all-in-one in vitroreaction. We engineered chassis strains that are optimized forcoordinated glycosylation and nsAA incorporation and produce up to 1-1.5g/L protein in cell-free protein synthesis. We showed that an orthogonaltranslation system (OTS) for incorporation of the p-azidophenylalanine(pAzF) nsAA can be functionally co-expressed with the nativeCampylobacter jejuni glycosylation pathway in the chassis strain andthat both OTS and glycosylation components are present in crude E. colilysates and participate in coordinated, in vitro pAzF incorporation andN-linked glycosylation. This resulted in the in vitro production ofpAzF-containing glycoproteins in reactions lasting 20 hours. Thistechnology has promising applications as a high-throughput prototypingplatform for glycoproteins containing bio-orthogonal chemical handlesthat are of potential biotechnological interest. This platform opens thedoor to on-demand synthesis of multiply modified glycoproteins forfundamental biology investigations or therapeutic applications

Applications

Applications of the disclosed technology include, but are not limitedto: (i) rapid expression of glycosylated and PEGylated proteins aspotential therapeutic candidate molecules; (ii) expression of multiplymodified proteins bearing one or more site-specifically installedpost-translational modifications for fundamental biology studies ortherapeutic applications; (iii) prototyping novel candidate moleculesfor PEGylated and glycosylated protein therapeutics; and (iv) productionplatform for long-acting glycosylated protein therapeutics

Advantages

Advantages of the disclosed technology include, but are not limited to:(i) first prokaryotic cell-free system capable of coordinated, cell-freetranscription, translation, non-standard amino acid incorporation, andglycosylation of proteins; (ii) enabled production of pAzF-containingglycoproteins in one-pot reactions lasting 20 hours; (iii) rapidprototyping of novel PEGylated or drug-conjugated glycoproteintherapeutics; and (iv) facile method for production of proteinscontaining multiple post-translational modifications (e.g.,glycosylation and phosphorylation) for fundamental biologyinvestigations or therapeutic applications.

Description of Technology

Attachment of chemical groups to proteins is typically accomplishedthrough conjugation chemistries with lysine or cysteine residues, orwith the N- or C-termini of the polypeptide backbone. However, theseapproaches typically result in heterogeneously conjugated proteinproducts, since the conjugation can take place at any of the reactiveresidues within a protein and can occur with varying degrees ofefficiency on the same protein substrate. To address this issue ofconjugate heterogeneity, nsAA incorporation has been shown to enablesite-specific conjugation of chemical groups to proteins through theintroduction of a bio-orthogonal chemical handle in the side chain ofthe nsAA.

Here, we leverage the specificity of nsAA incorporation and further addthe ability to glycosylate proteins site specifically. Others have shownthe ability to site-specifically incorporate a glycan into a protein viaenzymatic glycosylation of the introduction of an nsAA with aglycosylated side chain. Additionally, it has been shown that anexisting glycan on a protein can be chemically modified for attachmentof chemical groups. However, to our knowledge, there is no existingmethod for producing proteins containing both a bio-orthogonal chemicalhandle and a glycan. We show that this can be accomplished in a one-potin vitro reaction, opening the door to production of multiply modifiedproteins for fundamental investigations or therapeutic applications.

Our technology makes it possible to synthesize glycosylated proteinsbearing bio-orthogonal chemical handles in an all-in-one in vitroreaction. This advance decreases the time to produce these proteins fromdays to hours and has promising applications for the production ofglycoproteins modified with multiple chemical groups for fundamentalbiology studies (e.g., combined glycosylation and phosphorylation, etc.)or for therapeutic applications.

We are not are of any previously reported prokaryotic cell-free systemwith the capability to produce nsAA-containing glycoproteins. There arecommercial eukaryotic cell lysate systems for cell-free glycoproteinproduction (Promega, ThermoFisher), but these systems do not involveoverexpression of orthogonal glycosylation machinery and do not enablemodular, user-specified glycosylation in the manner that the presentlydisclosed system can. Additionally, to our knowledge, these systems havenot yet been shown to be compatible with nsAA incorporation, nor havethey been shown to enable coordinated nsAA incorporation andglycosylation.

There are commercial purified or prokaryotic crude lysate cell-freesystems for cell-free protein synthesis (Promega, ThermoFisher). Theseplatforms can be modified through the addition of purified OTScomponents for nsAA incorporation. However, unlike our engineeredstrains, the commercial crude lysate systems contain release-factor 1,which reduces the efficiency of nsAA incorporation. Additionally, noneof the commercial purified or prokaryotic cell-free systems have theability to carry out coordinated transcription, translation (includingnsAA incorporation), and glycosylation, which we describe here.

Our technology uniquely enables the facile, one-pot production ofproteins bearing both bio-orthogonal chemical handles and user-specifiedglycosylation. This type of molecule represents a very interesting andunexplored class of molecules that we anticipate will help advancefundamental science and have significant utility as a novel class oftherapeutic proteins. In particular, given the substantial currentinvestment from the biotechnology and pharmaceutical industries inprotein conjugates such as antibody-drug conjugates (ADCs), we think theability to synthesize glycoprotein conjugates will be of significantinterest to these industries in the future.

Example 2—Customizable, In Vitro Protein Glycosylation for Antibacterialand Anti-Cancer Vaccines

Reference is made to the presentation entitled “Customizable, in vitroprotein glycosylation for antibacterial and anti-cancer vaccines,” Starket al., presented on Apr. 17, 2018, at the American Association forCancer Research (AACR) Annual Meeting, and Abstract LB-304,Customizable, in vitro protein glycosylation for antibacterial andanti-cancer vaccines, Stark et al., published July 2018, CancerResearch, Volume 78, Issue 13, the contents of which are incorporatedherein by reference in their entireties.

Glycans represent a potential therapeutic antigen target. FIG. 1provides examples of tumor-associated glycans from Pochechueva et al.,Metabolites (2012) December; 2(4): 913-939, the content of which isincorporated herein by reference in its entirety. Particularlyillustrated in FIG. 1 are some of the glycans known to be involved ingynecological cancers. As such, the illustrated tumor-associated glycanmay be useful as therapeutic antigen targets against cancers such asgynecological cancers.

However, the inability to prepare protein carriers comprising preciseglycan structures limits the utility of glycans as therapeutic targets.For example, FIG. 2 illustrates the complexity of glycans observed inChinese Hamster Ovary (CHO) cells from Sheridan et al., Nat. Biotechnol.(2007), volume 25, pages 145-146, the content of which is incorporatedherein by reference in its entirety.

However, bacterial engineering enables programmable glycosylation inEscherichia coli. FIG. 3A illustrates the use of the Campylobacterjejuni N-glycosylation pathway in E. coli to prepare a recombinantglycosylated protein. (See Wacker et al., Science (2002), Nov. 29;298(5599):1790-3, the content of which is incorporated herein byreference in its entirety. FIG. 3B illustrates eukaryotic trimannosecore N-glycosylation in E. coli to prepare recombinant glycosylatedprotein. (See Valderrama-Rincon et al., Nat. Chem. Biol. (2012) Mar. 25;8(5):434-6. doi: 10.1038/nchembio.921, the content of which isincorporated herein by reference in its entirety.

Cell-free systems provide additional control over protein synthesis andglycosylation in vitro. FIG. 4 illustrates the use of cell-free biologyto enable precise control over protein synthesis and glycosylation.Cell-free systems offer an open reaction environment for facile additionor removal of glycosylation components and monitoring of reactionconditions.

FIG. 5 illustrates that crude E. coli lysates can be enriched withglycosylation components via overexpression in the source strain forlysate production. A CjLLO plasmid which expresses Campylobacter jejunilipid-linked oligosaccharides (LLO) and CjOST plasmid which expressesCampylobacter jejuni oligosaccharide transferases (OST) were introducedinto a source strain to provide a cell lysate enriched in LLO and OST.(See FIG. 5A). An acceptor plasmid expressing an acceptor polypeptidefor glycosylation was introduced into a cell-free glycoprotein synthesis(CFGpS) reaction mixture comprising the cell lysate to prepare aglycosylated acceptor polypeptide. (See FIG. 5A). FIG. 5B illustratesthat a glycosylated single chain antibody variable fragment (scFv) and aglycosylated green fluorescent protein (GFP) were synthesized in theCFGpS reaction mixture. Similarly, FIG. 6 illustrates that proteins suchas a single chain antibody variable fragment (scFv) can be glycosylatedwith bacterial and eukaryotic glycans using a cell-free glycoproteinsynthesis (CFGpS) platform.

Regarding glycan antigens, protein carriers typically are utilized tofor displaying the glycan antigens. Four proteins have been approved bythe US Food and Drug Administration (FDA) as adjuvant carriers forglycan antigens. These include Neisseria meningitides outer membraneprotein (PorA), Tetanus toxoid of Clostridium tetani, Diptheria toxoidof Corynebacterium diptheriae, Non-typeable Haemophilus influenzaederived protein D (hpd). (See FIG. 7 ).

FIG. 8 illustrates that protein carriers can be synthesized andglycosylated with Franciscella tularensis O antigen in vitro usingcell-free glycoprotein synthesis platforms. Crude E. coli lysates can beenriched with glycosylation components via overexpression in the sourcestrain for lysate production. FtLLO plasmid which expresses Franciscellatularensis lipid-linked oligosaccharides (LLO) including glycans formingthe Franciscella tularensis O antigen, and CjOST plasmid which expressesCampylobacter jejuni oligosaccharide transferases (OST) were introducedinto a source strain to provide a cell lysate enriched in LLO and OST.(See FIG. 8A). An acceptor plasmid expressing an FDA-approved proteincarrier was introduced into a cell-free glycoprotein synthesis (CFGpS)reaction mixture comprising the cell lysate to prepare a glycosylatedprotein carrier. (See FIG. 8A). FIG. 8B illustrates that carrierproteins (PD, PorA, CRM197, TTlight can be synthesized and glycosylatedwith Francisella tularensis O polysaccharide antibody in vitro using acell-free glycoprotein synthesis (CFGpS) platform.

Cell-free protein synthesis platforms can be configured to incorporatenon-standard amino acids. For example, FIG. 9 provides a schematicillustration of the use of amber suppression in a release factor 1(RF-1) deficient strain to incorporate a non-standard amino acid (nsAA).(See Perez, Stark, and Jewett, Cold Spring Harbor Perspect. Biol.(2016), Dec. 1; 8(12). pii: a023853. doi: 10.1101/cshperspect.a023853,the content of which is incorporated herein by reference in itsentirety). As illustarated in FIG. 9 , an orthogonal transfer RNA(o-tRNA) comprising the AUC anti-codon can be charged with a nsAA via anorthogonal aminoacyl-tRNA synthetase (o-aaRS). Elongation factor thermounstable (EF-Tu) then facilitates the selection and binding of thecharged o-tRNA to the A-site of the ribosome and the nsAA isincorporated in the nascent peptide chain at the position of the amber(UAG) codon. Because the strain is RF-1 deficient, translation is notterminated at the amber codon via competition between RF-1 and theEF-Tu/tRNA complex.

As contemplated herein, incorporation of a non-standard amino acid(nsAA) into a nascent protein and glycosylation of the nascent proteincan be combined in a cell-free glycoprotein synthesis (CFGpS) platform.FIG. 10 (top) schematically illustrates combined incorporation of anon-standard amino acid and glycan into a protein chain. The sfGFP-T216Xprotein was synthesized in vitro and the nsAA para-azido-phenylalaninewas incorporated into the protein. (See FIG. 10 (bottom left panel).Additionally, the sfGFP-T216X protein was glycosylated in vitro. (SeeFIG. 10 (bottom right panel).

In order to create improved cell lysates for CFGpS, the E. coli strainwas engineered to be deficient in in waaL or deficient in both of waaLand gmd. (See FIG. 11A). The E. coli waaL protein is associated withendogenous lipopolysaccharide synthesis and the E. coli gmd protein is aGDP-mannose 4,6-dehydratase associated with endogenous glycan synthesis.Therefore, a strain that is deficient in in waaL or deficient in both ofwaaL and gmd should produce a lysate having reduced components thatwould compete or interfere with orthogonal glycosylation. We observedthat cell lysates from the 705ΔwaaL strain and the 705ΔgmdΔwaaL strainwere efficient in CFGpS platforms. (See FIGS. 11A and 11B).

Using a cell lysate from the 705ΔwaaL strain we incorporated thenon-standard amino acid (nsAA) para-azido-phenylaline (pAzF) into thesfGFP T216X protein via cell-free protein synthesis. (See FIG. 12 ). Acell-free extract was prepare from the 705ΔwaaL strain and a purifiedorthogonal amino-acyl tRNA synthetase (aaRS) from Methanococcusjannaschii was added at increasing amounts to prepare CFPS reactionmixtures.

We next tested cell-free glycoprotein synthesis (CFGpS) of test proteins(scFV13-R4, sfGFP-T216X, and sfGFP-E132X) and incorporation ofnon-standard amino acid (nsAA) para-azido-phenylalanine (pAzF). (SeeFIG. 13 ). A cell-free extract was prepared from the 705ΔwaaL strain andtest conditions included: addition of para-azido-phenylalanine pAzF andorthogonal amino-acyl tRNA synthetase; addition of a cell free lysatecomprising the oligosaccharide transferase (OST) from Campylobacterjejuni; addition of a cell free lysate comprising the oligosaccharidetransferase (OST) from Campylobacter coli; addition of a cell freelysate comprising lipid-linked oligosaccharides (LLO) from Campylobacterjejuni; and presence of the glycosylation sequon DQNAT ornon-glycosylated variant AQNAT in the synthesized protein. The nsAA pAzFwas incorporated into each of scFV13-R4, sfGFP-T216X, and sfGFP-E132X.In addition, sfGFP-T216X, and sfGFP-E132X were observed to beglycosylated. (See FIG. 13 ).

Example 3—Toward Engineering Designer Glycoproteins with Site-SpecificFunctionalization Handles, and Editable Glycans

We generated glycoproteins that can be site-specifically labeled withuser-specified ligands and glycosylation patterns. Toward buildingeukaryotic-like glycosylation, we developed a system that can beinterfaced with bacterial glycosylation to site-specifically install thebase glycan for eukaryotic N-linked glycosylation, which can besubsequently elaborated by glycosyltransferases to build glycans ofchoice on a target protein.

The benefits of site-specific labeling of glycoproteins are vast. Forexample, numerous molecular therapeutics are labeled with polyethyleneglycol (PEG), as this modification has been attributed to boosts instability and half life of molecular therapeutics. Additionally,conjugating chemotherapeutic or antibiotic drugs to antibodies has beenshown to improve tumor or bacterial cell killing, respectively. Commonprotein labeling approaches, including the popular maleimide/cysteineand NHS-ester/lysine chemistries, have known drawbacks includingincomplete labeling and batch-to-batch heterogeneity. Additionally,non-specific labeling techniques can often affect the structure andfunction of folded protein molecules in unexpected ways.

To address these issues, we developed a system for combinedglycosylation and non-canonical amino acid (ncAA) incorporation thatwill enable site-specific labeling of glycoproteins via bio-orthogonalconjugation chemistries. Broadly, the site-specific incorporation ofncAAs is useful for increasing the chemical diversity of polypeptidesubstrates. This work leverages recent advances in synthetic biologywhich have led to the development of a suite of strains and orthogonaltranslation systems (OTSs) (FIG. 14A) that allow for ncAA incorporation(ncAAi), both inside of cells, and in cell-free, at high-fidelities andtiters. This technique, termed amber suppression refers to thereassignment of a native E. coli stop codon for the desired ncAA.Specifically for this work, we have focused on incorporating the ncAApara-azido-phenylalanine (pAzF), which is a partner in thestrain-promoted azide-alkyne cycloaddition (SPAAC) reaction (FIG. 14 ).This reaction is robust at a range of pH's and temperatures, and doesnot cross-react with any side groups found in the 20 canonical aminoacids (FIG. 14B).

We prepared CFGpS lysates using a chassis strain of E. coli that hasbeen genomically recoded for optimized glycosylation and ambersuppression (strain 705 ΔwaaL). Lysates were prepared from cellsoverexpressing the CjPglB enzyme and orthogonal pAzF tRNA (o-tRNA) witheither CjLLO or ClengLLO (FIG. 15 ). To test for ncAAi, we suppliedtemplate plasmids encoding sfGFP, bearing 0, 1, 3, or 5 amber codons. Inboth lysates, we observed production of sfGFP containing pAzF (FIG.15A). These lysates were then tested for glycosylation activity at threedifferent spike times, to test glycosylation efficiency over time asmore protein is synthesized (FIG. 15B). In both cases, we observed ˜100%glycosylation efficiency under all conditions tested.

We also developed methods for tailoring glycan structures on polypeptidesubstrates using enzymes to produce glycoproteins that are modified witha single GlcNAc for further elaboration (FIG. 16 ). Briefly, severalglycosylated polypeptides were incubated with an enzyme that trimsthrough poly GalNAc residues, run on a denaturing peptide gel, thenimaged vial in-gel florescence of an N-terminal fluorophore. The massshift of these peptides indicates that efficient trimming was achieved,leaving behind a truncated pentasaccharide when the substrate isglycosylated with a C. jejuni glycan, and a single GlcNAc residue whenthe glycan substrate is a linear glycan from C. lari (containing GlcNAcat the reducing end). These results have been confirmed by MALDI-MS. SeeFIG. 16B for expected oligosaccharide structures pre- and post-glycantrimming. In future work, we plan to combine our novel methods forncAAi, glycan trimming, and in vitro glycan building to producetherapeutic proteins bearing eukaryotic-like glycan structures andcontaining bio-orthogonal amino acids for site-specific chemicallabeling.

We also analyzed pAzF-incorporation in a cell-free glycoproteinsynthesis (CFGpS) system. First, we determined optimum concentrations ofof pAzF and pAzF-FS and measured background levels of incorporation in aCFGpS system by adding increasing amounts of pAzF and pAzF-FS. (See FIG.17 ). We next scaled-up CFGpS and purification. CFGpS reactions wereperformed at two different concentrations of aaRS, and purificationyields were 13.6 μM and 15.4 μM from 200 μl reaction volumes. (See FIG.17 ).

Finally, we performed cell-free glycoprotein synthesis (CFGpS) combinedwith strain-promoted alkyne-azide cycloadditions (SPAAC) to conjugate afluorophore to pAzF incorporated into a synthesized amino acid polymer.(See FIG. 18 ). SDS-PAGE analysis of purified, clicked productsindicated full conversion of pAzF-incorporated acceptor protein tofluorophore-labeled product. (See FIG. 18 ).

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PATENT DOCUMENTS

U.S. Pat. Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,518,058;6,783,957; 6,869,774; 6,994,986; 7,118,883; 7,189,528; 7,338,789;7,387,884; 7,399,610; 8,357,529; 8,574,880; 8,703,471; 8,999,668; and9,410,170; the contents of which are incorporated herein by reference intheir entirety.

U.S. Patent Publications: US20040209321; US20050170452; US20060211085;US20060234345; US20060252672; US20060257399; US20060286637;US20070026485; US20070154983; US20070178551; US20080138857;US20140295492; US20160060301; US20180016612; and US20180016614; thecontents of which are incorporated herein by reference in theirentirety.

Published International Applications: WO2003056914A1; WO2004013151A2;WO2004035605A2; WO2006102652A2; WO2006119987A2; WO2007120932A2;WO2014144583; and WO2017117539; the contents of which are incorporatedherein by reference in their entirety.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A cell-free glycoprotein synthesis (CFGpS) reaction mixturein a single reaction vessel comprising: (1) a cell lysate from agenomically recoded strain of Escherichia coli (E. coli) comprising: (a)a mutation in an endogenous (prfA) gene resulting in a deficiency theencoded release factor 1 protein; (b) a mutation in an endogenousO-antigen ligase (waaL) gene encoding an O-antigen ligase proteinresulting in a deficiency of the O-antigen ligase protein; (c) amutation in an endogenous DNA-specific endonuclease I (endA) geneencoding a DNA-specific endonuclease I protein resulting in a deficiencyof the endonuclease I protein; (d) a mutation in an endogenousGDP-mannose 4,6-dehydratase (gmd) gene encoding a GDP-mannose4,6-dehydratase protein resulting in reduced expression and/or activityof GDP-mannose 4,6-dehydratase as compared to wild-type E. coli; and (e)an orthogonal oligosaccharide transferase (OST), an orthogonal ligasefor synthesizing lipid-linked oligosaccharides (LLO), or both of anorthogonal oligosaccharide transferase (OST) and an orthogonal ligasefor synthesizing lipid-linked oligosaccharides (LLO); (2) a DNA templatefor expressing a sequence-defined amino acid polymer, a DNA-dependentRNA polymerase for transcribing an mRNA encoding the sequence-definedamino acid polymer, nucleotide triphosphates, amino acids, and an energysource; and (3) a non-standard amino acid (nsAA), an orthogonalamino-acyl tRNA synthetase (aaRS), or both of an nsAA and an aaRS,wherein the non-standard amino acid (nsAA) comprises a moiety thatreacts with a corresponding moiety on a saccharide to conjugate the nsAAto the saccharide; and (4) one or more components for performing astrain-promoted alkyne-azide cycloaddition (SPAAC) reaction.
 2. Thecell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1,wherein the strain is derived from Escherichia coli strain rEc.C321. 3.The cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim1, wherein the strain further comprises a mutation in a glutathionereductase (gor) gene encoding a glutathione reductase protein, resultingin a knock-out of the encoded glutathione reductase protein.
 4. Thecell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1,wherein the strain comprises a episomal or genomic vector for expressingan orthogonal oligosaccharide transferase (OST), an orthogonal ligasefor synthesizing lipid-linked oligosaccharides (LLO), or both of anorthogonal oligosaccharide transferase (OST) and an orthogonal ligasefor synthesizing lipid-linked oligosaccharides (LLO).
 5. The cell-freeglycoprotein synthesis (CFGpS) reaction mixture of claim 1 comprising anon-standard amino acid (nsAA), wherein the nsAA is selected frompara-azidophenylalanine (pAzF) and para-propargyloxy-phenylalanine(pAcF).
 6. The cell-free glycoprotein synthesis (CFGpS) reaction mixtureof claim 5, wherein the one or more components for performing the SPAACreaction comprise a dibenzocyclooctyne (DBCO) moiety.
 7. A method forpreparing a sequence defined amino acid polymer comprising: reacting thecell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1 toprepare the sequence defined amino acid polymer.
 8. The method of claim7, wherein the CFGpS reaction mixture comprises the non-standard aminoacid (nsAA) selected from para-azido-phenylalanine (pAzF) andpara-propargyloxy-phenylalanine (pAcF).
 9. The method of claim 8,wherein the one or more components for performing the strain-promotedalkyne-azide cycloaddition (SPAAC) reaction comprise adibenzocyclooctyne (DBCO) moiety for performing the SPAAC reaction withthe nsAA.